C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Der in US Patent 6,340,572 beschriebene Kit sieht die Anwendung eines Verdünnungsmittels für das biolumineszente Agens vor, dh für die Herstellung des Detektionsmittels werden dort zwei Komponenten und ein Verdünnungsschritt konzipiert. The kit described in US patent 6,340,572 provides for the application of a diluent for the bioluminescent agent before, ie two components and a dilution step, there designed for the production of the detection means.

Das in US Patent 6,017,722 beschriebene Verfahren weist die dort beschriebene Einschränkung hinsichtlich der Dauer der Biolumineszenz auf dem Chromatographiemedium auf. The method described in US Patent 6,017,722 has the limitation described there with respect to the duration of the bioluminescence on the chromatography medium.Diese Einschränkung folgt aus Verdunstungsprozessen, die die Vitalität der Mikroorganismen beeinträchtigen. This restriction follows from evaporation processes that affect the vitality of the microorganisms.

Der Träger mit den biologisch aktiven Substanzen kann eine Dünnschichtchromatographieplatte oder ein Elektrophoresegel oder ein anderes, bevorzugt planares, Trennsystem sein, auf dem sich die biologisch aktiven Substanzen in Form von Zonen befinden. The carrier with the biologically active substances can be a thin layer chromatography plate or an electrophoretic gel or some other, preferably planar, its separation system on which the biologically active substances in the form of zones are located.Die biologisch aktiven Substanzen können sich auch auf einem Träger in Form von Spots eines Substanzarrays befinden. The biologically active substances can also be located on a support in the form of spots of a substance array.

Die Detektion der Lumineszenz der Mikroorganismen-Suspension kann durch photographische Verfahren oder Imagingtechniken erfolgen. The detection of the luminescence of the microorganism suspension can be made by photographic methods or imaging techniques.

Die Beschichtung des Trägers kann durch Tauchen des Trägers in eine Suspension von Mikroorganismen erfolgen. The coating of the carrier can be effected by immersion of the support into a suspension of microorganisms.Während des Tauchvorgangs findet auf der zu beschichtenden Oberfläche eine Anreicherung der lumineszenten Mikroorganismen statt. During the immersion process an enrichment of the luminescent microorganisms takes place on the surface to be coated.So kann zB die Suspension von Vibrio fischeri aus einer Übernachtkultur bis auf das mehr als fünffache Volumen verdünnt werden und es wird immer noch die sichere Detektion toxischer Substanzen zB auf einer mit Kieselgel beschichteten Dünnschichtplatte erreicht. Thus the suspension of Vibrio fischeri from an overnight culture can be up to more than five times the volume of diluted, for example, and it is still the reliable detection of toxic substances achieved, for example on a surface coated with silica gel thin layer plate.Dieser Effekt ist überraschend, da man beim pH-Wert der Tauchsuspension (ca. pH 7) von einer negativen Ladung der Kieselgelmatrix und gleichzeitig einer negativen Oberflächenladung der Leuchtbakterien ausgehen muss. This effect is surprising since one (about pH 7) must be based on a negative charge of the silica matrix, while a negative surface charge of the luminescent bacteria at pH of diving suspension.Eine Erklärungsmöglichkeit für dieses unerwartete Verhalten bietet die Kompensation der elektrostatischen Kräfte durch andere Wechselwirkungen sowie eine reduzierte negative Ladungsdichte für die Bakterienhülle. A possible explanation for this unexpected behavior provides the compensation of the electrostatic forces by other interactions as well as a reduced negative charge density for the bacterial envelope.Messungen des Zetapotentials von Vibrio fischeri in der Tauchlösung stützen diese Annahme. Measurements of the zeta potential of Vibrio fischeri in the dipping solution support this assumption.

Durch das Tauchen erfolgt außerdem eine raschere Beschichtung des Trägers und es ergibt sich damit die Möglichkeit von zeitabhängigen Messungen bereits nach kurzen Einwirkungszeiten. By immersing also takes place a more rapid coating of the support and there is therefore the possibility of time-dependent measurements even after short exposure times.Auf diese Weise können akute toxische Effekte bereits innerhalb weniger Sekunden nachgewiesen werden. In this way acute toxic effects can already be detected within a few seconds.Weiterhin bleibt die Auftrennung von Substanzen auf dem Träger mit hoher Qualität erhalten, da in der kurzen Zeit kaum eine Diffusion der Substanzzonen stattfindet. Furthermore, the separation of substances on the carrier with high quality is maintained, since in the short time hardly any diffusion of the substance zones takes place.

Die biologische Aktivität kann, abhängig vom verwendeten biologischen System, sowohl eine Verringerung wie auch eine Zunahme der Lumineszenz bewirken. The biological activity can, depending on the used biological system, cause both a reduction as well as an increase in luminescence.In einem Messregime kann der zeitliche Verlauf der Inhibierung bzw. Stimulierung der Lumineszenz der Mikroorganismen-Suspension aufgezeichnet und ausgewertet werden. In a measurement regime of the time course of the inhibition or stimulating of the luminescence of the microorganism suspension can be recorded and evaluated.

Eine weitere Erhöhung der Lumineszenzintensität der Mikroorganismen und damit eine Verbesserung der Nachweisempfindlichkeit für die Detektion kann durch Kühlung der Mikroorganismen oder durch Lichteinstrahlung auf die Mikroorganismen erreicht werden. A further increase of the luminescence of the microorganisms and thus an improvement in the detection sensitivity for the detection can be achieved by cooling the microorganisms or by light irradiation on microorganisms.

Wird als Referenzsubstanz zB 4-Nitrophenol oder ein Kupfersalz verwendet, so werden als Prüfergebnis im Imagingsystem dunkle Zonen an den Positionen der Referenzsubstanzspots ausgewiesen. Is used as reference substance for example 4-nitrophenol or a copper salt, such dark areas are shown at the positions of the reference substance spots as the test result in the imaging system.Damit können sowohl die Funktion des mikrobiologischen Kits wie auch des für die eigentliche Messung notwendigen bildgebenden Systems validiert werden. Thus both the function of microbiological kits as well as the need for the actual measurement imaging system can be validated.

Claims (30)

Method for detecting biologically active substances comprising the steps of

a) providing a test support carrying substances to be tested, in particular a thin-layer chromatography plate, electrophoresis plate or a substance array,

b) providing a suspension containing microorganisms of the species Vibrio fischeri or a genetically modified variant of V. fischeri in a growth solution,

c) coating the test support with the microorganism suspension by dipping into the microorganism suspension,

d) detecting the biologically active substances on the test support by detecting change in luminescence of the microorganism suspension,

e) stimulating the luminescence of the microorganisms before or during detection, characterized in that the suspension contains an addition of aspartic acid in a concentration between 100 mg/l and 500 mg/l, and also in that

f) the luminescence of the microorganisms is stimulated by boron compounds, N-acylhomoserine lactones or biochemical precursor substances thereof or quinolones.

Method according to Claim 1, characterized in that the growth solution for the Vibrio fischeri suspension contains boron compounds, N-acylhomoserine lactones or biochemical precursor substances thereof or quinolones stimulating the luminescence of the microorganisms.

Method according to Claim 1 or 2, characterized in that the Vibrio fischeri suspension is obtained from freeze-dried microorganisms or frozen cell concentrates by reconstitution using a reconstitution medium.

Method according to one of Claims 1 to 3, characterized in that the Vibrio fischeri suspension is obtained by diluting a growth solution for the microorganisms by a dilution medium.

Method according to Claim 3 or 4, characterized in that the dilution medium or the reconstitution medium contains boron compounds, N-acylhomoserine lactones or biochemical precursor substances thereof or quinolones specifically inducing the luminescence of the microorganisms.

Method according to Claim 1 to 5, characterized in that a luminescence increase is further achieved by a high cell density of greater than 2 × 109 cells/ml in the Vibrio fischeri suspension.

Method according to Claim 1 to 6, characterized in that the support is coated homogeneously.

Method according to Claim 7, characterized in that, for coating the support, use is made of a dipping suspension, this being an up to more than five-fold dilution by volume of a suspension of an overnight culture of Vibrio fischeri.

Method according to Claim 1 to 8, characterized in that the luminescence of the Vibrio fischeri suspension is detected by photographic methods or imaging techniques.

Method according to Claim 1 to 9, characterized in that the biologically active substances have a stimulating or inhibiting effect on the luminescence of the Vibrio fischeri suspension.

Method according to Claim 10, characterized in that the time course of the inhibition or stimulating of the luminescence of the Vibrio fischeri suspension is recorded and analysed.

Method according to Claim 1 to 11, characterized in that the Vibrio fischeri suspension is subjected to cooling before and/or during the detection process.

Method according to Claim 1 to 12, characterized in that the Vibrio fischeri suspension is subjected to light irradiation before and/or during the detection process.

Kit for detecting biologically active substances on a test support, in particular a thin-layer chromatography plate, an electrophoresis plate, or a substance array containing Vibrio fischeri or a genetically modified variant of V. fischeri in a form stabilized for transport and storage, one or more growth media or the individual constituents for growth media for the microorganisms, characterized in that the growth medium contains aspartic acid in a concentration between 100 mg/l and 500 mg/l as addition for stimulating growth, and optionally boron compounds, N-acylhomoserine lactones or biochemical precursor substances thereof or quinolones as luminescence-stimulating substances.

Kit according to Claim 14, characterized in that the microorganisms were stabilized by freeze drying or by freezing together with suitable preservatives or by adsorption to suitable porous support materials or by inclusion in gel-like materials.

Kit according to Claim 14 or 15; characterized in that it contains a dipping chamber for the homogeneous coating of a support by the microorganisms.

Kit according to Claim 16, characterized in that the dipping chamber has a cooling jacket or heat exchanger for cooling the microorganism suspension.

Kit according to Claim 14 to 17, characterized in that it contains a test support to which defined amounts of reference substances having a known biological action have been applied.

Kit according to Claim 14 to 18, characterized in that the test support contains 4-nitrophenol or a copper salt as reference substance.

Kit according to Claim 14 to 19, characterized in that it further contains a device for the growth of microorganisms which contains a glass or plastic container.

Kit according to Claim 20, characterized in that the glass or plastic container, as a result of its design, ensures good oxygen exchange with the growth suspension and has a magnetic stirring bar.

Kit according to Claim 20 or 21, characterized in that the containers contain a growth solution and/or a dilution medium and/or a reconstitution medium.

Kit according to Claim 14 to 22, characterized in that the growth media contain boron compounds, N-acylhomoserine lactones or biochemical precursor substances thereof or quinolones which specifically induce the luminescence of the microorganisms.

Kit according to Claim 14 to 23, characterized in that it contains dilution media for diluting microorganism suspensions.

Kit according to Claim 24, characterized in that the dilution media contain aspartic acid in a concentration between 100 mg/l and 500 mg/l as addition for stimulating growth, and optionally boron compounds, N-acylhomoserine lactones or biochemical precursor substances thereof or quinolones as luminescence-stimulating substances.

Kit according to Claim 14 to 24, characterized in that it contains reconstitution media for producing ready-to-use microorganism suspensions from cell concentrates or stabilized preparations of microorganisms.

Kit according to Claim 26, characterized in that the reconstitution media contain aspartic acid in a concentration between 100 mg/l and 500 mg/l as addition for stimulating growth, and optionally boron compounds, N-acylhomoserine lactones or biochemical precursor substances thereof or quinolones as luminescence-stimulating substances.

Kit according to Claim 14 to 27, characterized in that it further contains a light source.

Kit according to Claim 14 to 28, characterized in that it further contains a cooling element or a heat exchanger for cooling the microorganism suspension.

Kit according to Claim 14 to 29, characterized in that it further contains a device for fractionating samples using planar separation techniques such as thin-layer chromatography or gel electrophoresis.